A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides

Abstract

An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin–Xa–GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.

Introduction

Protein purification is usually composed of multiple steps and typically regarded as a costly process in manufacture production. Various systems via fusion proteins produced by genetic engineering have been developed to simplify this process (Nygren et al., 1994, Ford et al., 1991). In general, a fusion protein expressed in these systems consists of an affinity tag, e.g., glutathione S-transferase, maltose-binding protein, cellulose-binding domain, polyhistidine, polycysteine, protein A, and streptavidin, covalently linked to the N- or C-terminus of the target protein (Smith and Johnson, 1998, Sakhamuru et al., 2000, Mateo et al., 2001, Terpe, 2003). To facilitate affinity purification, a specific ligand for the tag is immobilized onto a solid phase commonly packed in a column. After binding to the affinity column, the fusion protein of high purity is eluted either with excess ligand or by adjusting the pH of the buffer. Separation of the target protein from the tag is executed by limited proteolytic cleavage at their linker sequence either in the column or after elution (Schauer-Vukasionovic et al., 2002). Preparation and operation of these affinity columns are relatively simple but expensive.

Plant seed oil bodies are intracellular storage organelles for lipid preserved as a fuel for germination and subsequent seedling growth (Huang, 1996, Napier et al., 1996, Peng and Tzen, 1998, Frandsen et al., 2001, Murphy, 2001). An oil body of 0.5–2 μm in diameter contains a triacylglycerol (TAG) matrix surrounded by a monolayer of phospholipid (PL) and proteins (Chen et al., 1999, Lin et al., 2002). Oil bodies are remarkably stable both in cells and isolated preparation as a consequence of the steric hindrance and electronegative repulsion provided by the unique structural proteins, oleosins (Tzen and Huang, 1992, Tzen et al., 1992). According to the relative proportions of TAG, PL, and oleosin in native oil bodies, stable artificial oil bodies (AOB) can be technically constituted with these three essential constituents (Tzen and Huang, 1992, Tzen et al., 1998, Beisson et al., 2001, Tai et al., 2002, Peng et al., 2003).

Seed oil bodies extracted from transgenic plants expressing oleosin-fused proteins have been demonstrated to be adequate carriers for the production and purification of recombinant proteins (Van Rooijen and Moloney, 1995). On centrifugation, the target proteins fused with oleosins on the surface of oil bodies may be easily separated from the rest of the cellular extract as an oily ‘scum’, which floats on the top (Tzen et al., 1997). The resuspended oil bodies are either used directly as immobilized forms of target proteins or subjected to specific proteolytic cleavage at the designed linker sequence to separate target proteins from oleosins. Target proteins released from the recombinant oil bodies are harvested with high purity from the supernatant after one more centrifugation (Van Rooijen and Moloney, 1995). Operations involving recombinant oil bodies, in comparison with ligand affinity purification, are relatively inexpensive. However, the stability of introduced recombinant constructs and the expression level of recombinant proteins in transgenic plants should be evaluated prior to commercial application.

In this study, we developed a novel system to effectively produce recombinant proteins in Escherichia coli and easily purify the target proteins via AOB in a manner similar to the system using recombinant oil bodies from transgenic plants. A target protein, green fluorescent protein (GFP) was successfully expressed and purified with high quality and quantity.